Multiple and multivalent DNA vaccines in ovo

ABSTRACT

The present invention provides a muliple DNA vaccine and/or a multivalent DNA vaccine for use in aquiring embroyonic immunity in fowl eggs. The multiple DNA vaccine contains two or more DNA constructs, each containing a DNA molecule encoding an avian viral protein or a fragment thereof capable of inducing a protective immune response against the avian viral disease in fowl. The multivalent DNA vaccine contains one DNA construct which contains two or more DNA molecules, each representing an avian viral gene or a fragment thereof. The multivalent DNA vaccine is capable of expressing two or more viral antigens and inducing protective immune responses against the avian viral diseases in fowl. Both the multiple DNA vaccine and the multivalent DNA vaccine are preferred to be injected into the amniotic fluid of the fowl egg after being fertilized for about 18 days.

RELATED APPLICATION

This application claims the priority of U.S. Provisional ApplicationSer. No. 60/362,547, filed on Mar. 8, 2002, which is herein incorporatedby reference.

FIELD OF THE INVENTION

The present invention relates to either a muliple DNA vaccine or amultivalent DNA vaccine for use in aquiring embroyonic immunity in fowleggs and methods for preparing and using the same. The multiple DNAvaccine contains two or more DNA constructs, each containing a DNAmolecule encoding an avian viral protein or a fragment thereof capableof inducing a protective immune response against an avian viral diseasein fowl. The multivalent DNA vaccine contains a DNA construct whichcontains two or more DNA molecules. Each of the DNA molecules representsan avian viral gene or a fragment thereof. The multivalent DNA vaccineis capable of expressing two or more viral antigens and inducingprotective immune responses against two or more of the avian viraldiseases in fowl. Both the multiple DNA vaccine and the multivalent DNAvaccine are preferred to be injected into the amniotic fluid of the fowlegg after being fertilized for about 18 days.

BACKGROUND OF THE INVENTION

In ovo vaccination of virus-containing vaccines was extensivelydescribed by Sharma et al. (U.S. Pat. No. 4,458,630). In particular, itteaches that live Marek's disease virus can be injected into amnioticfluid within the egg, whereafter the embryo is infected and the vaccinevirus replicates to a high titer which induces the formation ofprotective antibodies in the treated embryo. (See Sharma (1985), AvianDiseases 29, 1155, 1167–68).

It is well-known in the worldwide poultry business that certain viraldiseases, such as Marek's disease virus (MDV), infectious bursal diseasevirus (IBDV), Newcastle disease virus (NDV), infectious bronchitis virus(IBV), infectious laryngotracheitis virus (ILTV), avianencephalomyelitis (AEV), chick anemia virus (CAV), Fowlpox virus (FPV),avian influenza virus (AIV), reovirus, avian leukosis virus (ALV),reticuloendotheliosis virus (REV), avian adenovirus and hemorrhagicenteritis virus (HEV), may cause major outbreak and result insignificant economic losses in the commercial poultry industry. Amongthem, MDV, IBDV, NDV and IBV, are particularly important due to theirvirulent nature.

Marek's Disease (MD) is a malignant, lymphoproliferative disorderdisease that occurs naturally in chickens. The disease is caused by aherpesvirus: Marek's Disease Virus (MDV). MD is ubiquitous, occurring inpoultry-producing countries throughout the world. Chickens raised underintensive production systems will inevitably suffer losses from MD. Thesymptoms of MD appear widely in the nerves, genital organs, internalorgans, eyes and skin of the infected birds, causing motor trouble (dueto paralysis when the nerves have been affected), functional trouble ofthe internal organs (due to tumors), and chronic undernourishment (ifthe internal organs are attacked by the virus). MD affects chickens fromabout 6 weeks of age, occurring most frequently between ages of 12 and24 weeks.

At of this time, there are no methods of treating MD. The control of thedisease is based primarily on management methods such as insolatinggrowing chickens from sources of infection, the use of geneticallyresistant stock, and vaccination. However, management procedures arenormally not cost-effective and the progress has been disappointing withrespect to the selection of poultry stock with increased geneticallycontrolled resistance. Nowadays, control of MD is almost entirely basedon vaccination.

Infectious bursal disease virus (IBDV) is responsible for a highlycontagious immunosuppressive disease in young chickens which causessignificant losses to the poultry industry worldwide (See Kibenge(1988), J. Gen. Virol., 69:1757–1775). Infection of susceptible chickenswith virulent IBDV strains can lead to a highly contagiousimmunosuppressive condition known as infectious bursal disease (IBD).Damage caused to the lymphoid follicles of the bursa of Fabricius andspleen can exacerbate infections caused by other agents and reduce achicken's ability to respond to vaccination as well (See Cosgrove(1962), Avian Dis., 6:385–3894).

IBDV is a member of the Birnaviridae family and its genome consists oftwo segments of double-stranded RNA (See Dobos et al (1979), J. Virol.,32:593–605). The smaller segment B (about 2800 bp) encodes VP 1, thedsRNA polymerase. The larger genomic segment A (about 3000 bp) encodes a110 kDa precursor polypeptide in a single open reading frame (ORF) thatis processed into mature VP2, VP3 and VP4 (See Azad et al (1985),Virology, 143:35–44). From a small ORF partly overlapping with thepolypeptide ORF, segment A can also encode VP5, a 17-kDa protein ofunknown function (See Kib

While VP2 and VP3 are the major structural proteins of the virion, VP2is the major host-protective immunogen and causes induction ofneutralizing antibodies (See Becht et al. (1988), J. Gen. Virol.,69:631–640; Fahey et al. (1989), J. Gen. Virol., 70:1473–1481). VP3 isconsidered to be a group-specific antigen because it is recognized bymonoclonal antibodies (Mabs) directed against VP3 from strains of bothserotype 1 and 2 (See Becht et al (1988), J. Gen. Virol., 69:631–640).(See Jagadish et al. (1988), J. Virol., 62:1084–1087).

In the past, control of IBDV infection in young chickens has beenachieved by live vaccination with avirulent strains, or principally bythe transfer of maternal antibody induced by the administration of liveand killed IBDV vaccines to breeder hens. Unfortunately, in recentyears, virulent variant strains of IBDV have been isolated fromvaccinated flocks in the United States (See e.g., Snyder et al. (1988),Avian Dis., 32:535–539; Van der Marel et al. (1990), Dtsch. Tierarztl.Wschr., 97:81–83), which drastically undermine the effectiveness ofusing live vaccination for IBDV.

Efforts to develop a recombinant vaccine for IBDV have also been made,and the genome of IBDV has been cloned (See Azad et al (1985)“Virology”, 143:35–44). The VP2 gene of IBDV has been cloned andexpressed in yeast (See Macreadie et al. (1990), Vaccine, 8:549–552), aswell as in recombinant fowlpox virus (See Bayliss et al (1991), Arch.Virol., 120:193–205). When chickens were immunized with the VP2 antigenexpressed from yeast, antisera afforded passive protection in chickensagainst IBDV infection. When used in active immunization studies, thefowlpox virus-vectored VP2 antigen afforded protection againstmortality, but not against damage to the bursa of Fabricius.

Newcastle disease virus (NDV) is an enveloped virus containing a linear,single-strand, nonsegmented, negative sense RNA genome. Typically, virusfamilies containing enveloped single-stranded RNA of the negative-sensegenome are classified into groups having non-segmented genomes (e.g.,Paramyxoviridae and Rhabdoviridae) or those having segmented genomes(e.g., Orthomyxoviridae, Bunyaviridae and Arenaviridae). NDV, togetherwith parainfluenza virus, Sendai virus, simian virus 5, and mumps virus,belongs to the Paramyxoviridae family.

The structural elements of the NDV include the virus envelope which is alipid bilayer derived from the cell plasma membrane. The glycoprotein,hemagglutinin-neuraminidase (HN) protrude from the envelope allowing thevirus to contain both hemagglutinin and neuraminidase activities. Thefusion glycoprotein (F), which also interacts with the viral membrane,is first produced as an inactive precursor, then cleavedpost-translationally to produce two disulfide linked polypeptides. Theactive F protein is involved in penetration of NDV into host cells byfacilitating fusion of the viral envelope with the host cell plasmamembrane. The matrix protein (M), is involved with viral assembly, andinteracts with both the viral membrane as well as the nucleocapsidproteins.

The main protein subunit of the NDV nucleocapsid is the nucleocapsidprotein (NP) which confers helical symmetry on the capsid. Inassociation with the nucleocapsid are the P and L proteins. Thephosphoprotein (P), which is subject to phosphorylation, is thought toplay a regulatory role in transcription, and may also be involved inmethylation, phosphorylation and polyadenylation. The L gene, whichencodes an RNA-dependent RNA polymerase, is required for viral RNAsynthesis together with the P protein. The L protein, which takes upnearly half of the coding capacity of the viral genome is the largest ofthe viral proteins, and plays an important role in both transcriptionand replication.

The replication of all negative-strand RNA viruses, including NDV, iscomplicated by the absence of cellular machinery required to replicateRNA. Additionally, the negative-strand genome can not be translateddirectly into protein, but must first be transcribed into apositive-strand (mRNA) copy. Therefore, upon entry into a host cell, thevirus can not synthesize the required RNA-dependent RNA polymerase. TheL, P and NP proteins must enter the cell along with the genome oninfection. Both the NDV negative strand genomes (vRNAs) and antigenomes(cRNAs) are encapsidated by nucleocapsid proteins; the onlyunencapsidated RNA species are virus mRNAs. The cytoplasm is the site ofNDV viral RNA replication, just as it is the site for transcription.Assembly of the viral components appears to take place at the host cellplasma membrane and mature virus is released by budding.

In U.S. Pat. No. 5,427,791, Ahmad et al. describe the embryonalvaccination against NDV, which requires the modification of the virusesthrough the use of ethyl methane sulfonate (EMS). However, EMS is amutagen so that the vaccine prepared by the use of EMS is suspected toact as a mutagen as well, which is undesirable for regularadministration of the vaccine. Nevertheless, without the modificationwith EMS, the NDV vaccine cannot be applied for in ovo vaccination asalmost all of the embryos will die upon injection of the eggs with theunmodified virus.

Infectious bronchitis virus (IBV), the prototype of the familyCoronaviridae, is the etiological agent of infectious bronchitis (IB).The virus has a single-stranded RNA genome, approximately 20 kb inlength, of positive polarity, and is usually about 80–100 nm in size,being round with projecting 20 nm spikes. IBV is the causative agent ofan acute, highly contagious disease in chickens of all ages, affectingthe respiratory, reproductive and renal systems.

IBV contains three structural proteins: the spike (S) glycoprotein, themembrane glycoprotein, and the nucleocapsid protein. The spikeglycoprotein is so called because it is present in the teardrop-shapedsurface projections or spikes protruding from the lipid membrane of thevirus. The spike protein is believed likely to be responsible forimmunogenicity of the virus, partly by analogy with the spike proteinsof other corona-viruses and partly by in vitro neutralisationexperiments (See, e.g., D. Cavanagh et al. (1984), Avian Pathology, 13,573–583). There are two spike glycoproteins, which are S1 (90,000daltons) and S2 (84,000 daltons). The polypeptide components of theglycopolypeptides S1 and S2 have been estimated after enzymatic removalof oligosaccharides to have a combined molecular weight of approximately125,000 daltons. It appears that the spike protein is attached to theviral membrane by the S2 polypeptide.

IBV has been wide-spread in countries where an intensive poultryindustry has been developed. Young chickens up to 4 weeks of age aremost susceptible to IBV, infection leading to high rates of morbidityand to mortality resulting from secondary bacterial infection. Infectionalso results in a drop in egg production, or failure to lay at fullpotential, together with an increase in the number of down-graded eggswith thin, misshapen, rough and soft-shells produced, which can have aserious economic effect.

Administering live vaccines to a developing chick in the egg (in-ovo)has proven to be a fast (40,000 eggs per hour), effective (100% of theeggs receive the vaccine), and labor saving ($100,000 per year perhatchery) method to vaccinate baby chicks against certain diseasesbefore they hatch.

The first in-ovo vaccination machine for use on chicken hatching eggswas developed by Embrex, Inc., of Raleigh, N.C. in the late 1980s. (SeeU.S. Pat. Nos. 5,056,464 and 5,699,751). This in-ovo machine iscurrently used in about 80% of the U.S. broiler hatcheries, primarilyfor administering MD vaccines. The popularity of this machine, which hasproven to be safe and effective in vaccination of chicks against MD, isalso being used increasingly to administer IBD vaccines and ND vaccines.

In the invention to be presented in the following sections, aDNA-mediated immunization (collectively “DNA vaccines”) will beintroduced. There are two kinds of DNA vaccines, i.e., a multiple DNAvaccine and a multivalent DNA vaccine. The multiple DNA vaccine of thepresent invention contains a combination of two or more DNA construct,each containing a single DNA molecule which is a viral gene or afragment thereof. The multivalent DNA vaccine of the present inventioncontains two or more viral genes or fragments thereof linking togetherin one DNA construct. The viral genes or fragments used in preparationof either the multiple DNA vaccine or the multivalent DNA vaccine arethose that encode viral peptides which are antigenic to and can induceboth the humoral and the cellular immune system in a host. The DNAvaccines are preferably applied to the egg by needles. The injection ofthe DNA vaccines in ovo leads to surprisingly strong immune responseswhich include not only antibody induction and T-cell activation withcytokine secretion, but also the production of cytotoxic T lymphocytes(CTL).

SUMMARY OF THE INVENTION

The present invention provides a multiple DNA vaccine for in ovoinjection. The multiple DNA vaccine contains two or more DNA constructs,each DNA construct expressing an antigenic protein of an avian viruscausing avian viral disease in fowl. The antigenic protein of the avianvirus is capable of inducing a protective immune response against anavian viral disease. The multiple DNA vaccine is preferred to injectinto the egg, particularly the amniotic fluid of the egg, of the fowl.The egg is preferred to be fertilized for about 18 days. The preferredfowl includes chicken, turkey, duck, and goose.

The DNA construct contains a DNA molecule and a vector. The vector canbe a plasmid or a viral carrier. The preferred vector is a plasmid.Examples of the plasmid include, but are not limited to, pcDNA3, pVAX1,pSectag, pTracer, pDisplay, pUC system plasmid (such as pUC7, pUC8,pUC18), and pGEM system plasmid. Alternatively, any plasmid whichcontains a promoter such as CMV promoter, SV40 promoter, RSV promoter,and β-actin promoter, can also be used for preparing the DNA construct.The most favorable plasmid is pcDNA3. The preferred viral carrier is oneselected from the group consisting of a baculovirus, a herpes virus, anda pox virus.

Examples of the avian virus include, but are not limited to Marek'sdisease virus (MDV), infectious vursal disease virus (IBDV), Newcastledisease virus (NDV), infectious bronchitis virus (IBV), infectiouslaryngotracheitis virus (ILTV), avian encephalomyelitis (AEV), avianleukosis virus (ALV), fowlpox virus (FPV), avian paramyxovirus (APV),duck hepatitis virus (DHV), and hemorrhagic enteritis virus (HEV).

The DNA molecules that are particularly suitable for inducing aprotective immune response against the avian viral diseases as shownabove include, but are not limited to, the entire of gB gene of Merk'sDisease virus (MDV) having the DNA sequence of SEQ ID NO:1 or a fragmentthereof; the entire VP2 gene of infectious bursal disease virus (IBDV)having the DNA sequence of SEQ ID NO:2 or a fragment thereof; the entireHN gene of Newcastle disease virus (NDV) having the DNA sequence (whichis from bases 6321 to 8319) of SEQ ID NO:3 or a fragment thereof (i.e.,SEQ ID NO:3 is the entire genome of the NDV); the entire S1 gene ofinfectious bronchitis virus (IBV) having the DNA sequence of SEQ ID NO:4or a fragment thereof; the entire glycoprotein G gene of infectiouslaryngotracheitis virus (ILTV) having the DNA sequence of SEQ ID NO:5 ora fragment thereof; the entire VP1, VP0, or VP3 gene of avianencephalomyelitis virus (AEV) or a fragment thereof (the VP1 gene hasthe DNA sequence of SEQ ID NO:6; the VP0 gene has the DNA sequence ofSEQ ID NO:7; and the VP3 gene has the DNA sequence of SEQ ID NO:8); theentire paraglycoprotein G gene of avian parainfluenza virus (APV) havingthe DNA sequence of SEQ ID NO:9 or a fragment thereof the entire type Apenton base gene of hemorrhagic enteritis virus (HEV) having the DNAsequence of SEQ ID NO:10 or a fragment thereof; and the entire envelopeantigen gene of fowlpox virus (FPV) having the DNA sequence of SEQ IDNO:11 or a fragment thereof.

One preferred example of the DNA vaccine contains two DNA constructs,each containing a DNA molecule capable of expressing a gene or afragment thereof which is from Marek's disease virus (MDV), infectiousvursal disease virus (IBDV), Newcastle disease virus (NDV), orinfectious bronchitis virus (IBV).

Another preferred example of the multiple DNA vaccine contains three ormore DNA constructs, each containing a DNA molecule capable ofexpressing a gene or a fragment thereof which is from Marek's diseasevirus (MDV), infectious vursal disease virus (IBDV), Newcastle diseasevirus (NDV), or infectious bronchitis virus (IBV).

The present invention also provides a method for vaccinating fowl eggand a method for preparing the multiple DNA vaccine. The method forvaccinating fowl egg includes injecting into the fowl egg the multipleDNA vaccine as shown above. The method for preparing the multiple DNAvaccine includes ligating a DNA molecule to a plasmid or virus carrierto form a DNA construct; and then mixing two or more said DNA constructsto form the multiple DNA vaccine. The insertion of the DNA molecule intothe vector can be achieved by conventional method, i.e., by ligation theDNA molecule with an enzyme such as T4 DNA ligase when both the genesand the desired vector have been cut with the same restriction enzyme(s)as complementary DNA termini are thereby produced. For pcDNA3, thepreferred restriction enzymes are BamH1 and EcoR1.

In another embedment, there is provided a multivalent DNA vaccine for inovo injection. The multivalent DNA vaccine comprises a DNA constructcontaining two or more DNA molecules linked together with a vector. Eachof the DNA molecules expresses an antigenic protein of an avian virus,which is capable of inducing a protective immune response against thatavian viral disease in fowl. The multivalent DNA vaccine is preferred tobe injected into a fowl egg. Each of the DNA molecules of themultivalent DNA vaccine is a gene or a fragment thereof from Marek'sdisease virus (MDV), infectious vursal disease virus (IBDV), Newcastledisease virus (NDV), infectious bronchitis virus (IBV), infectiouslaryngotracheitis virus (ILTV), avian encephalomyelitis (AEV), avianleukosis virus (ALV), fowlpox virus (FPV), avian parainfluenza virus(APV), duck hepatitis virus (DHV), and hemorrhagic enteritis virus(HEV).

DETAILED DESCRIPTION OF THE INVENTION

Traditional avian vaccines comprise chemically inactivated virusvaccines or modified live-virus vaccines. Inactivated vaccines requireadditional immunizations which are not only expensive to produce butalso laborious to administer. Further, some infectious virus particlesmay survive the inactivation process and may cause disease afteradministration to the animal.

In general, attenuated live virus vaccines are preferred overinactivated vaccines because they evoke an immune response often basedon both humoral and cellular reactions. Such vaccines are normally basedon serial passage of virulent strains in tissue culture. However, theattenuation process induces mutations of the viral genome, resulting ina population of virus particles heterogeneous with regard to virulenceand immunizing properties. In addition, it is well known that thetraditional attenuated live virus vaccines can revert to virulenceresulting in disease outbreaks in inoculated animals and the possiblespread of the pathogen to other animals.

Thus, it is advantageous for the industry to employ vaccines based onrecombinant DNA technology. The resulting DNA vaccines only contain andexpress the necessary and relevant immunogenic material that is capableof eliciting a protective immune response against the pathogens andwould not display above mentioned disadvantages of the live orinactivated vaccines.

For the purpose of preparing multiple DNA vaccines or multivalentrecombinant DNA vaccines, the DNA sequence of the gene (also usedinterchangeably as “DNA molecule”) need not contain the full length ofDNA encoding the polypeptides. In most cases, a fragment of the genewhich encodes an epitope region should be sufficient enough forimmunization. The DNA sequence of an epitope region can be found bysequencing the corresponding part of other viral strains and comparingthem. The major antigenic determinants are likely to be those showingthe greatest heterology. Also, these regions are likely to lieaccessibly in the conformational structure of the proteins. One or moresuch fragments of genes encoding the antigenic determinants can beprepared by chemical synthesis or by recombinant DNA technology. Thesefragments of genes, if desired, can be linked together or linked toother DNA molecules.

Also, the viral genes need not be in DNA. In fact, some of thefrequently found avian viral diseases are caused by double- orsingle-stranded RNA viruses. For example, Marek's diesease virus is adouble-stranded RNA virus, while infectious bursal disease virus (IBDV),Newcastle disease virus (NDV) and infectious bronchitis virus (IB) aresingle-stranded RNA viruses. The RNA viral sequences, however, can bereverse-transcribed into DNA using RT-Polymerase chain reaction (RT-PCR)technology and then incorporated into a vector by the conventionalrecombinant DNA technology

In addition, because of the degeneracy of the genetic code it ispossible to have numerous RNA and DNA sequences that encode a specifiedamino acid sequence. Thus, all RNA and DNA sequences which result in theexpression of a polypeptide having the antibody binding characteristicsare encompassed by this invention.

To construct a recombinant DNA vaccine, either univalent or multivalent,the DNA sequence of the viral gene can be ligated to other DNA moleculeswith which it is not associated or linked in nature. Optionally, the DNAsequence of a viral gene can be ligated to another DNA molecule, i.e., avector, which contains portions of its DNA encoding fusion proteinsequences such as β-galactosidase, resulting in a so-called recombinantnucleic acid molecule or DNA construct, which can be used fortransformation of a suitable host. Such vector is preferably derivedfrom, e.g., plasmids, or nucleic acid sequences present inbacteriophages, cosmids or viruses.

Specific vectors which can be used to clone nucleic acid sequencesaccording to the invention are known in the art and include either aplasmid or a virus carrier. Examples of the plasmid include, but are notlimited to, pBR322, pcDNA3, pVAX1, pSectag, pTracer, pDisplay, pUCsystem plasmids (e.g., pUC7, pUC8, pUC18), pGEM system plasmids,Bluescript plasmids or any other plasmids where CMV promoter, SV40promoter, RSV promoter, or β-actin promoter is included. The preferredplasmid is pcDNA3. Examples of the virus carrier include, but are notlimited to, bacteriophages (e.g., λ and the M13-derived phages), SV40,adenovirus, polyoma, baculoviruses, herpes viruses (HVT) or pox viruses(e.g., fowl pox virus).

The methods to be used for the construction of a recombinant nucleicacid molecule are known to those of ordinary skill in the art. Forexample, the insertion of the nucleic acid sequence into a cloningvector can easily be achieved by ligation with an enzyme such as T4 DNAligase when both the genes and the desired cloning vehicle have been cutwith the same restriction enzyme(s) so that complementary DNA terminiare thereby produced.

Alternatively, it may be necessary to modify the restriction sites so asto produce blunt ends either by digesting the single-stranded DNA or byfilling in the recessive termini with an appropriate DNA polymerase.Subsequently, blunt end ligation with an enzyme such as T4 DNA ligasemay be carried out. If desired, any restriction site may be produced byligating linkers onto the DNA termini. Such linkers may comprisespecific oligonucleotide sequences that encode restriction sitesequences. The restriction enzyme cleaved vector and nucleic acidsequence may also be modified by homopolymeric tailing.

The present invention provides two kinds of DNA vaccines. The first kindis a multiple DNA vaccine, which includes two or more of univalent DNAvaccines, each containing a DNA sequence encoding at least onepolypeptide affording protection against one viral disease such asMarek's dosease voris (MDV), infectious bursal disease virus (IBDV),Newcastle disease virus (NDV), infectious bronchitis virus (IBV),infectious laryngotracheitis virus (ILTV), avian encephalomyelitis(AEV), Fowlpox virus (FPV), avian influenza virus (AIV), avian leukosisvirus (ALV), duck hepatitis virus B genome, and hemorrhagic enteritisvirus (HEV), inserted into a commercially available plasmid.

The second kind is a multivalent recombinant DNA vaccine, which containstwo or more genes or gene fragments from different viruses. These genesor gene fragments are carried by a useful vector, which can be either aplasmid or a virus carrier. The multivalent recombinant DNA vaccineencodes two or more antigenic polypeptides which afford protectionagainst at least two viral diseases including, but not limited to, MD,IBD, ND or IB. The viral genes or gene fragments are operativelyattached to the vector in reading frame so that they can be expressed ina host. The different structural DNA sequences carried by the vector maybe separated by termination and start sequences so that the proteins canbe expressed separately or they may be part of a single reading frameand therefore be produced as a fusion protein by methods known in theart.

The preferred DNA sequences include, but are not limited to, the entireof gB gene of Merk's Disease virus (MDV) having the DNA sequence of SEQID NO:1 or a fragment thereof; the entire VP2 gene of infectious bursaldisease virus (IBDV) having the DNA sequence of SEQ ID NO:2 or afragment thereof; the entire HN gene of Newcastle disease virus (NDV)having the DNA sequence of SEQ ID NO:3 or a fragment thereof; the entireS1 gene of infectious bronchitis virus (IBV) having the DNA sequence ofSEQ ID NO:4 or a fragment thereof.

The DNA sequence encoding the gB polypeptide of MDV has the nucleic acidsequence as SEQ ID NO:1. The DNA sequence contains 3650 bp of linearDNA.

The DNA sequence encoding the VP2 polypeptide of IBDV has the nucleicacid sequence as SEQ ID NO:2. The DNA sequence contains 3004 bp oflinear DNA molecule which is reversely transcribed from IBDV's RNAtemplate.

The DNA sequence of the entire genome of NDV contains 15186 bps of DNA,wherein (1) base No. 56 to 1792 encodes NP polypeptide, which isnucleocapsid protein; (2) base No. 1804–3244 encodes P polypeptide,which is a phosphoprotein; (3) base No. 3256–4487 encodes M polypeptide,which is a matrix protein; (4) base No. 4498–6279 encodes F polypeptide,which is a fusion protein; (5) base 6321–8319 encodes HN polypeptide,which is a hemagglutinin-neuraminidase; (6) base No. 8370–15073 encodesL polypeptide, which is a large polymerase protein. The NDV genome hasthe DNA sequence as SEQ ID NO:3.

The DNA sequence of the Si polypeptide contains 1611 bp of linear DNAsequence as shown in SEQ ID NO:4, which is reversely transcribed fromIBV's RNA templates.

The following experimental designs are illustrative, but not limitingthe scope of the present invention. Reasonable variations, such as thoseoccur to reasonable artisan, can be made herein without departing fromthe scope of the present invention.

I. Materials and Methods

(A) Virus and Vaccines

Avian infectious bronchitis virus (IBV), infectious bursal disease (IBD)and Newcastle disease (ND) vaccines were purchased from Intervet Inc.

(B) Viral RNA Isolation and RT-PCR

Two hundred microliter recovered attenuated vaccines (Intervet Inc.)were resolved in iced cold GTC buffer (4 M guanidium isothiocyanate, 25mM sodium citrate, pH 7.0, 0.5% Sarkosyl, 0.1 M-mercaptoethanol) andsodium acetate (pH 4). An equal volume of phenol-chloroform (1:1) wasadded and placed on ice for 15 minutes after vortexing. The aqueousphase was collected after centrifuge and the RNA was precipitated withan equal volume of isopropanol. RNA was pelleted by centrifugation at12000 rpm for 20 min at 4° C. and then suspended in diethylpyrocarbonate(DEPC) treated deionized distill water and stored at −70° C.

(C) Oligonucleotides

Oligonucleotide primers for RT-PCR amplification were purchased fromPromega, and were designed according to the genome of the Avianinfectious bronchitis virus (Beaudette CK strain), Newcastle diseasevirus (Lasota strain) and Infectious bursa disease virus respectively.The sequences of the primers used for PCR were:

IBS1F′ (SEQ ID NO:12) 5′ CGGGATCCGCCGCCGCCATGTTGGTAACACCTCTT 3′; IBS1R′(SEQ ID NO:13) 5′ CGGAATTCTTAACGTCTAAAACGACGTGT 3′; NDF F′ (SEQ IDNO:14) 5′ CGGGATCCGCCGCCGCCATGGGCTCCAGACCTTCTACC 3′; NDF R′ (SEQ IDNO:15) 5′ CCGCTCGAGTTACATTTTTGTAGTGGCTCTCATT 3′; NDHN F′ (SEQ ID NO:16)5′ CGGGATCCGCCGCCGCCATGGACCGCGCCGTTAGGCAAG 3′; NDHN R′ (SEQ ID NO:17)5′ GCTCTAGATTACTCAACTAGCCAGACCTG 3′; IBDVP2F′ (SEQ ID NO:18)5′ CGGGATCCGCCGCCGCCATGACAAACCTGCAAGAT 3′; IBDVP2R′ (SEQ ID NO:19)5′ CGGAATTCTTACCTTATGGCCCGGATTAT 3′.(D) Reverse Transcription Polymerase Reaction (RT-PCR)

Reverse transcription of IBV, NDV and IBDV RNA were carried out at 42°C. for 30 min in 2.5× Taq buffer (200 mM NaCl, 15 mM Tris-HCl, pH7.4, 15mM MgCl₂, 15 mM β-mercaptoethanol, and 0.25 mM each of dATP, dCTP, dGTP,and dTTP). In addition to the Taq buffer, the reaction mixture (40 μl)also contained viral RNA, 2.4 U of avian myeloblastosis virus (AMV)reverse transcriptase (Promega), 16 U of RNasin (Promega), and 0.01 nmolreverse primer (IBDVP2R, NDF F, NDHN F or IBS1R). The final volume ofthe reaction mixture was 40 μl. After reverse transcription, thefollowing reagents were added to the reverse transcription mixture: 0.02nmol of each nucleotide triphosphate (dATP, dCTP, dGTP, dTTP), 0.01 nmolof forward primer (IBDVP2F, NDF R, NDHN R or IBS1F) and 1.5 U of Taq DNApolymerase (Strategene). Water was then added to a final volume of 100μl. The reaction was carried out for 32 cycles in a Thermal Cycler(Perkin Elmer-Cetus). Each PCR cycle consisted of 1 min of denaturationat 94° C., 1 min of annealing at 57° C., and 2 min of DNA chainelongation at 72° C.

(E) Preparation of DNA Constructs

The plasmids pCMV-VP2, pCMV-S1, pCMV-NDF and pCMV-NDHN were constructedwith the VP2, S1, NDF and NDHN genes from IBD vaccine, IBV vaccine andNDV vaccine respectively, placed downstream of the commercial plasmidpcDNA3. (Invitrogen, U.S.A.). All of the genes were inserted into thepcDNA3 vector using restriction enzymes BamH1, EcoR1, XbaI and XhoI(underlined characters in the sequence of the primers). Sequences of theall genes in the pcDNA3 vector were verified by sequencing in bothdirections.

(F) Preparation of DNA and DNA Delivery

The quantity of plasmid DNA that had been purified by affinitychromatography (Qiagen. Inc.) was determined by spectrophotometricmeasurements at 260 and 280 nm. The DNA in aliquots to 100 μg wassuspended in 100 μl of PBS (0.14M NaCl, 10 mM sodium phosphate, pH 7.4).For DNA delivery, 1 cc syringe with a 20 gauge 1 and ½ inch needle wereused. For the in-ovo groups, the embryos (18-day-old fertilized anddeveloping eggs from the setting trays) were injected with 0.1milliliters of DNA vaccine (100 μg) into the large end of each eggthrough the air cell with a needle. The eggs were then transferred intothe hatchery where they remained until they hatched at about 21 days ofage. For the IM (Intramuscular), all of the vaccines (⅕ dose of livevaccines) were injected into the chicken's thoracic muscle at 10 dayspost hatchery.

II. Experimental Design

Specific Pathogen Free (SPF) fertilized eggs (n=60) were randomized into12 groups. All groups (five eggs each group), all eggs were given 100 μlin volume each. 100 μg pCMV-NDF+100 μg pCMV-NDHN mixture was injected ineach egg of group A, 100 μg pCMV-S1 was injected in each egg of group B,100 μg pCMV-VP2 was injected in each egg of group C, 100 μg pCMV-NDF+100μg pCMV-NDHN+100 μg pCMV-S1(ND+IB) was injected in each egg of group D,100 μg pCMV-NDF+100 μg pCMV-NDHN+100 μg pCMV-VP2 (ND+IBD) was injectedin each egg of group E, 100 μg pCMV-VP2+100 μg pCMV-VP2 mixture (IB+IBD)was injected in each egg of group F, 100 μg pCMV-NDF+100 μgpCMV-NDHN+100 μg pCMV-S1+100 μg pCMV-VP2 mixture (ND+IB+IBD) wasinjected in each egg of group G, one dose of commercialized in-ovo IBDvaccine (Embrex, Inc) was injected in each egg of group H as positivecontrol, 100 ul PBS was injected in each egg of group I, J, K and L. Allchickens in this experiment were given 100 μl in volume (⅕ dose of livevaccines), injected into the chicken's thoracic muscle each at 10 dayspost hatchery. Chickens in group A and I were injected with NDV vaccine,group B and J were injected with IBV vaccine, group C and K wereinjected with IBDV vaccine, group D were injected with the mixture ofNDV+IB vaccines, group E were injected with the mixture of NDV+IBDvaccines, group F were injected with the mixture of IB+IBD vaccines andgroup G and L were injected with the mixture of NDV, IB and IBDvaccines.

III. Serology Detection

All of the serum samples were collected at 10 days (injected with lowdose live vaccines at the same time), 17 days, 24 days and 31 days posthatchery. The antibody titers were detected by ELISA using IB, IBD andNDV antibody test kits which purchased from IDEXX Laboratories, Inc. Allof the samples were detected duplicated. Dilute test samples fivehundred fold (1:500) with sample diluents prior to being assayed. Thetest procedure was applied according to the kit's manual. For the assayto be valid, measure and record absorbance values at 650 nm, A (650).The relative level of antibody in the unknown was determined bycalculating the sample to positive (S/P) ratio. Endpoint titers werecalculated using the formula: Log₁₀Titer=1.09(Log₁₀S/P)+3.36

Results

As shown in Table 1, the results demonstrated that, for the detection ofanti-IBD antibodies, the IBDV recombinant antigens VP2 could beexpressed and played the role of primary stimulation. The titersincreased rapidly after a low dose vaccine booster. The titers of groupC, E, F and G at 17 days post hatchery (i.e. 7 days post IM injection)were significantly higher than those of group K and L. Most importantly,the expression of IBDV antigen was not interfered by other monovalentDNA vaccines (NDV and IBV). The same results were also applied to IB andNDV DNA vaccines. The titers of group B, D, F and G were higher thanthose of group J and L at 17 days post hatchery (Table 2) and the titersof group A, D, E and G were higher than those of group I and L at 17days post hatchery (Table 3). The only unpredicted result was theanti-NDV titer could not be highly induced by the triple valent DNAvaccine (Table 3, group G), but anti-IBD and anti-IB did (Tables 1 and2, group G).

TABLE 1 Serum Samples Detected by IDEXX IBD Antibody Test Kit (Ab TitersCorrespond to the Average Titers ±SD) Immunization and Sample CollectionSchedule (days) Animal Group 10 Days PH* 17 Days PH 24 Days PH 31 DaysPH C(IBD)  —** 4535 ± 1267 16623 ± 3105 21254 ± 3852 E(IBD + ND) — 1685± 655  17339 ± 2185 19041 ± 2967 F(IBD + IB) — 8252 ± 2205 10057 ± 129517561 ± 2006 G(IBD + IB + ND) — 9111 ± 1701 13127 ± 1763 16694 ± 2134H(IBD positive) 6553 ± 851 13025 ± 2131  18015 ± 1592 18853 ± 2614K(PBS/IBD) — — 1853 ± 302 17002 ± 2965 L(PBS/IBD + IB + ND) — —  6923 ±1168 18063 ± 2531 *PH: post hatchery **—: average titers less than 396(be considered negative by IDEXX kit)

TABLE 2 Serum Samples Detected by IDEXX IB Antibody test kit (Ab TitersCorrespond to the Average Titers ±SD Immunization and Sample CollectionSchedule (days) Animal Group 10 Days PH* 17 Days PH 24 Days PH 31 DaysPH B(IB)  —** 441 ± 117 2426 ± 264 3214 ± 877 D(IB + ND) — 586 ± 182 805 ± 221 1988 ± 501 F(IB + IBD) — 509 ± 89   685 ± 186 1192 ± 237G(IBD+ IB + — 499 ± 81  688 ± 78 2551 ± 531 ND) J(PBS/IB) — — 485 ± 761662 ± 441 L(PBS/IBD + — —  819 ± 202 1332 ± 488 IB + ND) *PH: posthatchery **—: average titers less than 396 (be considered negative byIDEXX kit)

TABLE 3 Serum Samples Detected by IDEXX ND Antibody Test Kit (Ab TitersCorrespond to the Average Titers ±SD). Immunization and SampleCollection Schedule (days) Animal Group 10 Days PH* 17 Days PH 24 DaysPH 31 Days PH A(ND) — 466 ± 101 2394 ± 456 8103 ± 2198 D(ND + IB) — 706± 140 1778 ± 378 6811 ± 2206 E(ND + IBD) — 517 ± 104 3021 ± 411 5991 ±1695 G(IBD + IB + — — — 783 ± 201 ND) I(PBS/ND) — — 1853 ± 324 3912 ±304  L(PBS/IBD + — — 4027 ± 662 5807 ± 1996 IB + ND) *PH: post hatchery**—: average titers less than 396 (be considered negative by IDEXX kit)

1. A method for vaccinating a fowl egg comprising: injecting into anamniotic fluid of said fowl egg a multiple DNA vaccine, the multiple DNAvaccine comprising: two or more DNA constructs, each of said DNAconstructs containing a DNA molecule and a vector; wherein said DNAmolecule comprises a gene or a fragment thereof encoding an antigenicpeptide of an avian virus causing an avian viral disease in fowl;wherein said antigenic peptide of said avian virus is capable ofinducing a protective immune response against said avian viral diseasein said fowl; wherein said vector is a plasmid or a viral carrier;wherein said viral carrier is selected from the group consisting of abacteriophage, an SV40, an adenovirus, a polyoma virus, a vacciniavirus, a baculovirus, and a pox virus; and wherein said avian virus isselected from the group consisting of Marek's disease virus (MDV),infectious bursal disease virus (IBDV), Newcastle disease virus (NDV),infectious bronchitis virus (IBV), infectious laryngotracheitis virus(ILTV), avian leucosis virus (ALV), duck hepatitis virus (DHV), andhemorrhagic enteritis virus (HEV).
 2. A method for vaccinating a fowlegg comprising: injecting into an amniotic fluid of said fowl egg amultivalent DNA vaccine, said multivalent DNA vaccine comprising: a DNAconstruct containing two or more DNA molecules ligated to a vector,wherein each of said DNA molecules contains a gene or a fragment thereofencoding an antigenic peptide of an avian virus causing an avian viraldisease in fowl; wherein said antigenic peptide of said avian virus iscapable of inducing a protective immune response against said avianviral disease in said fowl; wherein said vector is a plasmid or a viralcarrier; wherein said viral carrier is selected from the groupconsisting of a bacteriophage, an SV40, an adenovirus, a polyoma virus,a vaccinia virus, and a baculovirus; and wherein said avian virus isselected from the group consisting of Marek's disease virus (MDV),infectious bursal disease virus (IBDV), Newcastle disease virus (NDV),infectious bronchitis virus (IBV), infectious laryngotracheitis virus(ILTV), avian leucosis virus (ALV), duck hepatitis virus (DHV), andhemorrhagic enteritis virus (HEV).
 3. A method for delivering a multipleDNA vaccine to a fowl, the method comprising: injecting said multipleDNA vaccine into an amniotic fluid of an egg of said fowl, the multipleDNA vaccine comprising: two or more DNA constructs, each of said DNAconstructs containing a DNA molecule and a vector; wherein said DNAmolecule comprises a gene or a fragment thereof encoding an antigenicpeptide of an avian virus causing an avian viral disease in fowl;wherein said antigenic peptide of said avian virus is capable ofinducing a protective immune response against said avian viral diseasein said fowl; wherein said vector is a plasmid or a viral carrier;wherein said viral carrier is selected from the group consisting of abacteriophage, an SAV40, an adenovirus, a polyoma virus, a vacciniavirus, a baculovirus, and a pox virus; and wherein said avian virus isselected from the group consisting of Marek's disease virus (MDV),infectious bursal disease virus (IBDV), Newcastle disease virus (NDV),infectious bronchitis virus (IBV), infectious laryngotracheitis virus(ILTV), avian leucosis virus (ALV), duck hepatitis virus (DHV), andhemorrhagic enteritis virus (HEV).
 4. The method according to claim 3,wherein said DNA vaccine is injected into a large end of said eggthrough an air cell with a needle.
 5. The method according to claim 4,wherein said needle is a 20 gauge 1 and ½ inch needle.
 6. A method fordelivering a multivalent DNA vaccine to a fowl, the method comprising:injecting said multivalent DNA vaccine into an amniotic fluid of an eggof said fowl, said multivalent DNA vaccine comprising: a DNA constructcontaining two or more DNA molecules ligated to a vector, wherein eachof said DNA molecules contains a gene or a fragment thereof encoding anantigenic peptide of an avian virus causing an avian viral disease infowl; wherein said antigenic peptide of said avian virus is capable ofinducing a protective immune response against said avian viral diseasein said fowl; wherein said vector is a plasmid or a viral carrier;wherein said viral carrier is one selected from the group consisting ofa bacteriophage, an SV40, an adenovirus, a polyoma virus, a vacciniavirus, and a baculovirus; and wherein said avian virus is one selectedfrom the group consisting of Marek's disease virus (MDV), infectiousbursal disease virus (IBDV), Newcastle disease virus (NDV), infectiousbronchitis virus (IBV), infectious laryngotracheitis virus (ILTV), avianleucosis virus (ALV), duck hepatitis virus (DHV), and hemorrhagicenteritis virus (HEV).
 7. The method according to claim 6, wherein saidDNA vaccine is injected into a large end of said egg through an air cellwith a needle.
 8. The method according to claim 7, wherein said needleis a 20 gauge 1 and ½ inch needle.
 9. A method for vaccinating a fowlegg comprising: injecting into an amniotic fluid of said fowl egg amultivalent DNA vaccine, said multivalent DNA vaccine comprising: a DNAconstruct containing two or more DNA molecules ligated to a vector,wherein each of said DNA molecules contains a gene or a fragment thereofencoding an antigenic peptide of an avian virus causing an avian viraldisease in fowl; wherein said antigenic peptide of said avian virus iscapable of inducing a protective immune response against said avianviral disease in said fowl; wherein said vector is a plasmid; andwherein said avian virus is one selected from the group consisting ofMarek's disease virus (MDV), infectious bursal disease virus (IBDV),Newcastle disease virus (NDV), infectious bronchitis virus (IBV),infectious laryngotracheitis virus (ILTV), avian leucosis virus (ALV),duck hepatitis virus (DHV), and hemorrhagic enteritis virus (HEV).
 10. Amethod for delivering a multivalent DNA vaccine to a fowl, the methodcomprising: injecting said multivalent DNA vaccine into an amnioticfluid of an egg of said fowl, said multivalent DNA vaccine comprising: aDNA construct containing two or more DNA molecules ligated to a vector,wherein each of said DNA molecules contains a gene or a fragment thereofencoding an antigenic peptide of an avian virus causing an avian viraldisease in fowl; wherein said antigenic peptide of said avian virus iscapable of inducing a protective immune response against said avianviral disease in said fowl; wherein said vector is a plasmid; andwherein said avian virus is one selected from the group consisting ofMarek's disease virus (MDV), infectious bursal disease virus (IBDV),Newcastle disease virus (NDV), infectious bronchitis virus (IBV),infectious laryngotracheitis virus (ILTV), avian leucosis virus (ALV),duck hepatitis virus (DHV), and hemorrhagic enteritis virus (HEV). 11.The method according to claim 1, wherein said DNA vaccine is injectedinto a large end of said egg through an air cell with a needle.
 12. Themethod according to claim 1, wherein the vector is a plasmid.
 13. Themethod according to claim 1, wherein the fowl egg is a chicken egg. 14.The method according to claim 1, wherein the fowl egg is a turkey egg.15. The method according to claim 1, wherein the egg is fertilized forabout 18 days.
 16. The method according to claim 2, wherein said DNAvaccine is injected into a large end of said egg through an air cellwith a needle.
 17. The method according to claim 2, wherein the fowl eggis a chicken egg.
 18. The method according to claim 2, wherein the fowlegg is a turkey egg.
 19. The method according to claim 2, wherein theegg is fertilized for about 18 days.
 20. The method according to claim3, wherein the vector is a plasmid.
 21. The method according to claim 3,wherein the fowl is a chicken.
 22. The method according to claim 3,wherein the fowl is a turkey.
 23. The method according to claim 3,wherein the egg is fertilized for about 18 days.
 24. The methodaccording to claim 6, wherein the fowl is a chicken.
 25. The methodaccording to claim 6, wherein the fowl egg is a turkey egg.
 26. Themethod according to claim 6, wherein the egg is fertilized for about 18days.